Legionella pneumophila is a protypical example of an emerging infectious disease threat. Recent exciting data from several labs have indicated that type I interferons (IFNs) are an important signature of infection with many, if not all, intracellular pathogens. Our hypothesis is that a cytosolic surveillance pathway detects Legionella-derived ligands in the cytosol leading to the transcriptional induction of type I IFNs and coregulated genes. Preliminary data indicate that type I interferons are induced by Legionella in a manner dependent on L. pneumophila's type IV (Dot/Icm) secretion system. We also find that type I interferons are required to restrict intracellular replication of Legionella. The putative Legionella-derived ligand that stimulates host production of interferon is unknown, but our preliminary studies have identified Mda5 as a key host sensor of L. pneumophila. Since Mda5 is a cytosolic sensor of RNA, our finding suggests the exciting possibility that L. pneumophila may translocate bacterial RNA into the host cell cytosol. Even if a non-RNA ligand from L. pneumophila is sensed by Mda5, our results challenge existing paradigms since Mda5 is widely believed to be solely a sensor of viruses, rather than of bacteria. Thus, our specific aims are: 1. Identify and characterize molecular determinants of L. pneumophila that positively or negatively regulate host production of type I interferon. 2. Characterize the host pathways that sense L. pneumophila in the cytosol, leading to transcriptional induction of type I interferon and other genes. 3. Determine the role of cytosolic sensing and type I interferons in innate immunity against L. pneumophila using in vitro and in vivo models. PUBLIC HEALTH RELEVANCE: Legionella pneumophila is a bacterium that replicates in host cells called macrophages, thereby causing a severe, and often lethal, pneumonia called Legionnaires'Disease. In this proposal we seek to use Legionella as a model for understanding how macrophages sense and defend against infection, with the ultimate goal of using this information to develop more effective therapeutics and vaccines.